A. Field of the Invention
The invention generally concerns the field of protein engineering. More particularly, disclosed herein are variants of T7 RNA polymerase with the ability to incorporate modified nucleotides and with enhanced transcriptional activity.
B. Description of Related Art
RNA is widely versatile and useful, but its chemical instability can render it unsuitable for many therapeutic and biotechnology functions. Oligonucleotides with altered chemistry, especially modifications of 2′ position of the (deoxy)ribose have proven to be of great value (Wilson & Keefe, 2006). 2′-O-methyl RNA has a greater Tm, faster kinetics, and greater stability as antisense probes (Majlessi, et al., 1998) and siRNA with 2′F and 2′-O-methyl RNA have also proven to be more stable and target-specific (Layzer, 2004; Kraynack & baker, 2006; Jackson, et al., 2006; Dean & Bennet, 2003. Additionally, in vitro selection with 2′ modified NTPs has yielded aptamers and ribozymes with greater stability and enhanced chemical potential (Healy, et al., 2004; Waters, et al., 2011; Lupold, et al., 2002; Keefe & Cload, 2008; Burmeister, et al., 2005; Beaudry, et al., 2000).
While modified RNA can be chemically synthesized it is often preferable to enzymatically produce it (especially for in vitro selection) (Ellington & Szostak, 1990). T7 RNA polymerase has long been utilized for the generation of RNA in vitro, and has previously been engineered and evolved to have an expanded substrate range. Most famously, the Y639F mutant allows for the polymerization of RNA transcripts contain nucleotides with 2′-Fluoro and 2′-amino modified ribose (Kostyuk, et al., 1995; Sousa & Padilla, 1995; Huang, et al., 1997). A further mutation, H784A, is thought to eliminate premature termination following the incorporation of a modified nucleotide, and the Y639F, H784A (“FA”) double mutant can incorporate nucleotides with bulky modifications at the 2′ position (e.g. 2′-O-methyl)(Padilla & Sousa, 2002; Brieba & Sousa, 2004).
A directed evolution approach, in which the aforementioned Y639 and H784 residues, as well as the important R425 and G542 were randomized, has been previously employed to create further T7 RNA polymerase variants with expanded substrate specificity (Chelliserrykattil & Ellington, 2004). The resulting library was enriched for T7 RNA polymerase variants that retained the ability to transcribe RNA in vivo (with natural ribose) and the screened for altered substrate specificities in vitro. A mutant, termed “RGVG,” (R425, G542, Y639V, H784G plus additional E593G and V685A mutations that arose organically during the selection) showed strong activity with 2′-O-methyl UTP. A second mutant, termed “VRS,” (G542V and H784S as well as the additional H772R mutation) was able to incorporate 2′-Fluoro modified pyrimidines. More recent works have also uncovered the “2P16” mutant (a version of RGVG with seven additional mutations (Siegmund, et al., 2012)) and the R425C mutant (Ibach, et al., 2013). Each of these mutants is reported to enable the creation of 2′-O-methyl RNA.
While the unique catalytic properties of these enzymes make them useful tools, several of them suffer from low activity, even with normal ribonucleotides. It has been proposed that mutations that confer new activity in an enzyme also destabilize the protein, rendering it less active overall, with low transcriptional yields (Wang, et al., 2002; Romero, et al., 2009).